Establishment Of Genetic Transformation System For Epicotyl And Cotyledon From Seedlings Of ‘Fengmiguan' And Agrobacterium-Mediated Transformation Of Bt Gene

Jujube which has the characteristics of drought resistance and barren resistance is adaptable is one of the important economic tree species in China.Jujube fruit is popular by its good nutritional quality.Traditional breeding technology of jujube,such as grafting,was greatly limited the development of jujube cultivation because of the heavy workload and long breeding cycle.Meanwhile,common pests,such as jujube inchworm,jujube armyworm,jujube carposina niponensis walshingham and cnidocampa flavescens also greatly restricted the production of jujube cultivation.With the expansion of jujube cultivation area and the spread and increasing of pests,the research of jujube pests is imminent.Modern molecular biotechnology,especially the development of plant tissue culture and genetic engineering,provides important technical support for directional improvement and new variety breeding of Chinese jujube.While transgenic technology makes it possible for rapidly creating jujube insect-resistant germplasm.The effects of different pre-culture duration,Infection time and co-culture duration,different concentration of bacterium solution and AS,and different infection mode on the GUS transient expression were compared by on Agrobacterium-Mediated transformation of ‘Fengmiguan' using epicotyls and cotyledons as explants.Then the Bt gene was transferred into ‘Fengmiguan' by using the optimized genetic transformation system,which laid a foundation for cultivating insect-resistant germplasm.The main results are as follows:1.Kan sensitivity experiment of different explantsThe effects of different concentration of Kan on callus formation and adventitious bud regeneration were explored using the epicotyls and cotyledons as explants.The results indicated that 50 mg/L Kan was the optimal concentration of inhibiting the regeneration of epicotyls and cotyledons.2.Establishment of genetic transformation system for epicotyl and cotyledon of ‘Fengmiguan'The effects of different pre-culture duration,infection time and co-culture duration,different concentration of bacterium solution and AS,and different infection mode on transformation efficiency were compared through GUS transient expression rate.The results showed that the optimal treatment of epicotyl was 0.5 of bacteria solution concentration OD600,10 min of vacuum assisted infection time,3 d of co-culture duration and adding 20 mg/L AS into co-culture medium.While the optimal treatment of cotyledon was 3 d of pre-culture duration,0.5 of bacteria solution concentration OD600,10 min of vacuum assisted infection time,3 d of co-culture duration and adding 10 mg/L AS into co-culture medium.3.Agrobacterium-mediated Transformation of Bt Gene for different explants of ‘Fengmiguan'Bt gene was transferred into ‘Fengmiguan' by Agrobacterium-mediated transformation using three different explants.The transformation ability of three explants was compared.21 of PCR-positive shoots were obtained through PCR analysis.